Clinical studies and studies on isolated islets or Beta cell lines have indicated that chronic exposure of the Beta cell to supraphysiological levels of glucose results in impaired Beta cell function, an important component in the pathogenesis of type 2 diabetes. How glucose exerts this toxicity upon the Beta cell remains unclear. We propose that the glucose metabolite, glucosamine plays a role in Beta cell function and this glucose toxicity. We came to this hypothesis as a result of our observations on the mechanism of toxicity of the Beta cell- specific toxin, streptozotocin (STZ). STZ is chemically analogous to N- acetylglucosamine (GlcNAc). Furthermore, we found that the Beta cell contains approximately 100-fold more of the mRNA encoding the enzyme o- GlcNAc transferase (OGT). This enzyme modifies nuclear and cytoskeletal proteins by linking the monosaccharide GlcNAc to serine or threonine residues in the protein. The resulting O-GlcNAc modification appears to modify the activity of transcription factors. We found that STZ blocks the activity of an enzyme that removes o-GlcNAc from proteins. Treatment of rats with STZ results in the accumulation of the O-GlcNAc modification specifically in the pancreatic Beta cells, hours before Beta cell death. Because the Beta cells are so richly endowed with OGT, these cells may be the most susceptible to an accumulation of O-GlcNAc on intracellular proteins. We have also shown in other cell types, that nuclear O-GlcNAc is sensitive to ambient glucose concentrations. If this is also true in the Beta cell, then hyperglycemia and STZ may both increase Beta cell O-GlcNAc content, thereby leading to a common mechanism of Beta cell toxicity. The experiments proposed in this grant are designed to determine if hyperglycemia indeed result in increased Beta cell O-GlcNAc. We will also create transgenic mouse models in which glucosamine synthesis from glucose is either augmented or decreased. We will determine the effect of these alterations in glucosamine metabolism on Beta cell function. We have also found that cAMP-dependent protein kinase inhibits the enzyme responsible for glucosamine synthesis. We propose to study the mechanism by which glucosamine synthesis is inhibited. Together, these studies will establish the role of glucosamine in glucose toxicity on the Beta cell and a means of controlling glucose metabolism to glucosamine.